A bilayer cell‐free protein synthesis system for high‐throughput screening of gene products

FEBS Letters

Tsuchimochi, Masateru¹  Endo, Yaeta¹  Hasegawa, Yoshinori¹  Kuroita, Toshihiro²  Ogasawara, Tomio¹  Sawasaki, Tatsuya¹  Kamura, Nami¹ 

[1] Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan;Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., Toyo-cho, Tsuruga 914-0047, Japan

关键词: Cell-free protein synthesis;    Bilayer reaction method;    Escherichia coli coupled transcription–translation;    Wheat germ system;    High-throughput screening;    CBB;    Coomassie brilliant blue;    FAD;    flavin adenine dinucleotide;    GFP;    green fluorescent protein;    KOD;    archaeon Pyrococcus sp. strain KOD1 DNA polymerase;    ER;    estrogen receptor protein;   

DOI  :  10.1016/S0014-5793(02)02329-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.

【 授权许可】

Unknown